TY - JOUR
T1 - The effect of 24R,25-(OH)2D3 on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1α,25-(OH)2D3 is mediated by phospholipase C
AU - Schwartz, Z.
AU - Sylvia, V. L.
AU - Luna, M. H.
AU - DeVeau, P.
AU - Whetstone, R.
AU - Dean, D. D.
AU - Boyan, B. D.
N1 - Funding Information:
The authors wish to thank Sandra Messier and Virginia Contreras for their help in preparing the manuscript and David Lopez and Teresa Guinee for their technical assistance. We also thank Dr. Anthony Norman (University of California, Riverside, CA) for his generous gift of 24S,25-(OH) 2 D 3 . This study was supported by US PHS Grants DE-05937 and DE-08603 and the Center for the Enhancement of the Biology/Biomaterials Interface (CEBBI) at the University of Texas Health Science Center at San Antonio.
PY - 2001
Y1 - 2001
N2 - 1α,25-(OH)2D3 regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)2D3 regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)2D3 and determined the role of phospholipases D, C, and A2 as well as G-proteins in mediating the effects of vitamin D3 metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)2D3. Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)2D3. This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)2D3 on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1α,25-(OH)2D3-dependent PKC. Inhibition of PI-PLC blocked the 1α,25-(OH)2D3-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA2 with melittin inhibited basal and 24R,25-(OH)2D3-stimulated PKC in RC cells and stimulated basal and 1α,25-(OH)2D3-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)2D3 on PKC, whereas GDPβS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1α,25-(OH)2D3 regulates PKC in GC cells via PI-PLC and PLA2, but not PC-PLC or PLD, whereas 24R,25-(OH)2D3 regulates PKC in RC cells via PLD2.
AB - 1α,25-(OH)2D3 regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)2D3 regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)2D3 and determined the role of phospholipases D, C, and A2 as well as G-proteins in mediating the effects of vitamin D3 metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)2D3. Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)2D3. This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)2D3 on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1α,25-(OH)2D3-dependent PKC. Inhibition of PI-PLC blocked the 1α,25-(OH)2D3-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA2 with melittin inhibited basal and 24R,25-(OH)2D3-stimulated PKC in RC cells and stimulated basal and 1α,25-(OH)2D3-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)2D3 on PKC, whereas GDPβS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1α,25-(OH)2D3 regulates PKC in GC cells via PI-PLC and PLA2, but not PC-PLC or PLD, whereas 24R,25-(OH)2D3 regulates PKC in RC cells via PLD2.
KW - 24R,25-(OH)D
KW - Cell maturation
KW - Chondrocyte cultures
KW - Phospholipase D
KW - Protein kinase C
KW - Signal transduction
UR - http://www.scopus.com/inward/record.url?scp=0034858752&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034858752&partnerID=8YFLogxK
U2 - 10.1016/S0039-128X(01)00100-3
DO - 10.1016/S0039-128X(01)00100-3
M3 - Article
C2 - 11546556
AN - SCOPUS:0034858752
SN - 0039-128X
VL - 66
SP - 683
EP - 694
JO - Steroids
JF - Steroids
IS - 9
ER -